1. Technical Field
The subject invention relates to the identification of a gene involved in the elongation of long-chain polyunsaturated fatty acids (i.e., xe2x80x9celongasexe2x80x9d) and to uses thereof. In particular, elongase is utilized in the conversion of one fatty acid to another. For example, elongase catalyzes the conversion of gamma linolenic acid (GLA) to dihomogamma linolenic acid (DGLA). Elongase also catalyzes the conversion of stearidonic acid (18:4n-3) to (n-3)-eicosatetraenoic acid (20:4n-3). DGLA, for example, may be utilized in the production of other polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA) which may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.
2. Background Information
The elongases which have been identified in the past differ in terms of the substrates upon which they act. Furthermore, they are present in both animals and plants. Those found in mammals have the ability to act on saturated, monounsaturated and polyunsaturated fatty acids. In contrast, those found in plants are specific for saturated or monounsaturated fatty acids. Thus, in order to generate polyunsaturated fatty acids in plants, there is a need for a PUFA-specific elongase.
The elongase is, in fact, a four-enzyme complex. In both plants and animals, the elongation process is the result of this four-step mechanism (Lassner et al., The Plant Cell 8:281-292 (1996)). CoA is the acyl carrier. Step one involves condensation of malonyl-CoA with a long-chain acyl-CoA to yield carbon dioxide and a xcex2-ketoacyl-CoA in which the acyl moiety has been elongated by two carbon atoms. Subsequent reactions include reduction to xcex2-hydroxyacyl-CoA, dehydration to an enoyl-CoA, and a second reduction to yield the elongated acyl-CoA. The initial condensation reaction is not only the substrate-specific step but also the rate-limiting step.
As noted previously, elongases, more specifically, those which utilize PUFAs as substrates, are critical in the production of long-chain polyunsaturated fatty acids which have many important functions. For example, PUFAs are important components of the plasma membrane of a cell where they are found in the form of phospholipids. They also serve as precursors to mammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins. Additionally, PUFAs are necessary for the proper development of the developing infant brain as well as for tissue formation and repair. In view of the biological significance of PUFAs, attempts are being made to produce them, as well as intermediates leading to their production, efficiently.
A number of enzymes are involved in PUFA biosynthesis including elongases (elo) (see FIG. 1). For example, linoleic acid (LA, 18:2-xcex949,12 or 18:2n-6) is produced from oleic acid (18:1-xcex949) by a xcex9412 desaturase. GLA (18:3-xcex946,9,12) is produced from linoleic acid by a xcex946-desaturase. AA (20:4-xcex945,8,11,14) is produced from dihomo-xcex3-linolenic acid (DGLA, 20:3-xcex948,11,14) by a xcex945-desaturase. As noted above, DGLA is produced from GLA by elongase.
It must be noted that animals cannot desaturate beyond the xcex949 position and therefore cannot convert oleic acid into linoleic acid. Likewise, xcex1-linolenic acid (ALA, 18:3-xcex949,12,15) cannot be synthesized by mammals. However, xcex1-linolenic acid can be converted to stearidonic acid (STA, 18:4-6,9,12,15) by a xcex946-desaturase (see PCT publication WO 96/13591; see also U.S. Pat. No. 5,552,306), followed by elongation to (n-3)-eicosatetraenoic acid (20:4-xcex948,11,14,17) in mammals and algae. This polyunsaturated fatty acid (i.e., 20:4-xcex948,11,14,17) can then be converted to eicosapentaenoic acid (EPA, 20:5-xcex945,8,11,14,17) by a xcex945-desaturase. Other eukaryotes, including fungi and plants, have enzymes which desaturate at carbons 12 (see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and 15 (see PCT publication WO 93/11245). The major polyunsaturated fatty acid of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid or xcex1-linolenic acid. In view of these difficulties, it is of significant interest to isolate genes involved in PUFA biosynthesis from species that naturally produce these fatty acids and to express these genes in a microbial, plant or animal system which can be altered to provide production of commercial quantities of one or more PUFAs. Consequently, there is a definite need for the elongase enzyme, the gene encoding the enzyme, as well as recombinant methods of producing this enzyme. Additionally, a need exists for oils containing levels of PUFA beyond those naturally present as well as those enriched in novel PUFAs. Such oils can only be made by isolation and expression of the elongase gene.
One of the most important long chain PUFAs, noted above, is arachidonic acid (AA). AA is found in filamentous fungi and can also be purified from mammalian tissues including the liver and the adrenal glands. As noted above, AA production from is catalyzed by a xcex945-desaturase, and DGLA production from xcex3-linolenic acid (GLA) is catalyzed by an elongase. However, until the present invention, no elongase had been identified which was active on substrate fatty acids in the pathways for the production of long chain PUFAs and, in particular, AA and EPA or 20:5n-3.
Two genes appeared to be of interest in the present search for the elongase gene. In particular, the jojoba xcex2-ketoacyl-coenzyme A synthase (KCS), or jojoba KCS, catalyzes the initial reaction of the fatty acyl-CoA elongation pathway (i.e., the condensation of malonyl-CoA with long-chain acyl-CoA (Lassner et al., The Plant Cell 8:281-292 (1996)). Jojoba KCS substrate preference is 18:0, 20:0, 20:1, 18:1, 22:1, 22:0 and 16:0. Saccharomcyes cerevisiae elongase (ELO2) also catalyzes the conversion of long chain saturated and monounsaturated fatty acids, producing high levels of 22:0, 24:0, and also 18:0, 18:1, 20:0, 20:1, 22:0, 22:1, and 24:1 (Oh et al., The Journal of Biological Chemistry 272 (28):17376-17384 (1997); see also U.S. Pat. No. 5,484,724 for a nucleotide sequence which includes the sequence of ELO2; see PCT publication WO 88/07577 for a discussion of the sequence of a glycosylation inhibiting factor which is described in Example V). The search for a long chain PUFA-specific elongase in Mortierella alpina began based upon a review of the homologies shared between these two genes.
The present invention includes an isolated nucleotide sequence corresponding to or complementary to at least about 50%, preferably at least about 60%, and more preferably at least about 70% of the nucleotides in sequence from the nucleotide sequence shown in SEQ ID NO:1 (FIG. 6), and fragments thereof. The isolated nucleotide sequence may be represented by SEQ ID NO:1. All of the above sequences may encode a functionally active elongase which utilizes a polyunsaturated fatty acid as a substrate. These sequences may be derived from a fungus of the genus Mortierella and may be of the species alpina. Additionally, the present invention includes a purified protein encoded by any of the nucleotide sequences described above, as well as a purified polypeptide which elongates polyunsaturated fatty acids and has at least about 50% amino acid similarity to the amino acid sequence of the purified protein.
Furthermore, the present invention includes a method of producing elongase enzyme comprising the steps of: a) isolating the nucleotide sequence represented by SEQ ID NO:1 (FIG. 6); b) constructing a vector comprising: i) the isolated nucleotide sequence operably linked to ii) a promoter; and c) introducing the vector into a host cell under time and conditions sufficient for expression of the elongase enzyme. The host cell may be selected from the group consisting of a eukaryotic cell or a prokaryotic cell. The prokaryotic cell may be selected from the group consisting of E. coli, cyanobacteria, and B. subtilis. The eukaryotic cell may be selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell. The fungal cell may be a yeast cell such as, for example, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida spp., Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. or Pichia spp. Preferably, Saccharomyces cerevisiae is utilized.
The present invention also includes a vector comprising: a) a nucleotide sequence as represented by SEQ ID NO:1 (FIG. 6) operably linked to b) a promoter. Furthermore, the invention also includes a host cell comprising this vector. Again, the host cell may be selected from the group consisting of a eukaryotic cell or a prokaryotic cell. The prokaryotic cell may be selected from the group consisting of E. coli, cyanobacteria, and B. subtilis. The eukaryotic cell may be selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell. The fungal cell may be, for example, a yeast cell. The yeast cell may be selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida spp., Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. and Pichia spp. Preferably, the host cell is Saccharomyces cerevisiae. 
Additionally, the present invention includes a recombinant plant cell, plant or tissue comprising the vector described above, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid by the plant cell, plant or tissue. The polyunsaturated fatty acid may be, for example, selected from the group consisting of GLA and STA.
The invention also includes one or more plant oils expressed by the recombinant plant cell or plant tissue.
Also, the present invention includes a transgenic plant comprising the vector described above, wherein expression of said nucleotide sequence of the vector results in production of a polyunsaturated fatty acid in seeds of the transgenic plant.
Furthermore, the present invention also encompasses a transgenic, non-human mammal whose genome comprises a DNA sequence encoding an elongase operably linked to a promoter. This DNA sequence may be represented by SEQ ID NO:1 (FIG. 6). The invention also includes a fluid produced by the transgenic, non-human mammal wherein the fluid comprises a detectable level of at least one elongase and/or a detectable level of metabolites formed by the activity of the above-described elongase(s) (i.e., an altered level of, for example, DGLA, eicosatetraenoic acid (20:4n-3), AA or EPA).
Additionally, the present invention includes a method for producing a polyunsaturated fatty acid comprising the steps of: a) isolating the nucleotide sequence represented by SEQ ID NO:1 (FIG. 6); b) constructing a vector comprising the isolated nucleotide sequence; c) introducing the vector into a host cell under time and conditions sufficient for expression of the elongase enzyme; and d) exposing the expressed elongase enzyme to a xe2x80x9csubstratexe2x80x9d polyunsaturated fatty acid in order to convert the substrate to a xe2x80x9cproductxe2x80x9d polyunsaturated fatty acid. The substrate polyunsaturated fatty acid may be, for example, GLA or STA and the product polyunsaturated fatty acid may be, for example, DGLA or 20:4n-3, respectively.
A second method may further comprise the step of exposing the expressed elongase enzyme to a desaturase in order to convert the product polyunsaturated fatty acid to another polyunsaturated fatty acid. The product polyunsaturated fatty acid may be, for example, DGLA or 20:4n-3, the xe2x80x9canotherxe2x80x9d polyunsaturated fatty acid may be, for example, AA or EPA, respectively, and the desaturase may be, for example, xcex945-desaturase. The second method may further comprise the steps of exposing the xe2x80x9canotherxe2x80x9d polyunsaturated fatty acid to the elongase and an additional desaturase in order to convert the another polyunsaturated fatty acid to a xe2x80x9cfinalxe2x80x9d polyunsaturated fatty acid (i.e., a third method). This final polyunsaturated fatty acid may be, for example, docosahexaenoic (DHA) acid.
Additionally, the present invention includes a nutritional composition comprising at least one polyunsaturated fatty acid selected from the group consisting of a product polyunsaturated fatty acid produced according to the first method, another polyunsaturated fatty acid produced according to the second method, and a final polyunsaturated fatty acid produced according to the third method. The product polyunsaturated fatty acid may be, for example, DGLA or 20:4n-3. The another polyunsaturated fatty acid may be, for example, AA or EPA. The final polyunsaturated fatty acid may be, for example, DHA. The nutritional composition may be, for example, selected from the group consisting of an infant formula, a dietary supplement and a dietary substitute, and may be administered to a human or to an animal. The composition may be administered enterally or parenterally and may further comprise at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, monoglycerides, borage oil, diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and protein hydrolysates. It may further comprise at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex and at least one mineral selected from the group consisting of calcium magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium and iron.
The present invention also encompasses a pharmaceutical composition comprising 1) at least one polyunsaturated fatty acid selected from the group consisting of the product polyunsaturated fatty acid produced according to the first method, another polyunsaturated fatty acid produced according to the second method, and the final polyunsaturated fatty acid produced according to the third method and 2) a pharmaceutically acceptable carrier. The composition may be administered to a human or an animal. It may further comprise an element selected from the group consisting of a vitamin, a mineral, a carbohydrate, an amino acid, a free fatty acid, a phospholipid, an antioxidant, and a phenolic compound.
Additionally, the present invention includes an animal feed comprising at least one polyunsaturated fatty acid selected from the group consisting of the product polyunsaturated fatty acid produced according to the first method, another polyunsaturated fatty acid produced according to the second method and a final polyunsaturated fatty acid produced according to the third method. The product polyunsaturated fatty acid may be, for example, DGLA or 20:4n-3. The another polyunsaturated fatty acid may be, for example, AA or EPA. The final polyunsaturated fatty acid may be, for example, DHA.
The present invention also includes a cosmetic comprising a polyunsaturated fatty acid selected from the group consisting of a product polyunsaturated fatty acid produced according to the first method, another polyunsaturated fatty acid produced according to the second method and a final polyunsaturated fatty acid produced according to the third method.
Additionally, the present invention also encompasses a method of preventing or treating a condition caused by insufficient intake of polyunsaturated fatty acids comprising administering to the patient the nutritional composition described above in an amount sufficient to effect treatment.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.